Upregulation of programmed death ligand-1 in tumor-associated macrophages affects chemotherapeutic response in ovarian cancer cells

To better understand the mechanism of chemoresistance in ovarian cancer cells, we aimed to investigate the influence of macrophages on the tumor cell response to carboplatin and identify the genes associated with chemoresistance. We mimicked the tumor microenvironment (TME) using a co-culture technique and compared the proliferation of ovarian cells with and without macrophages. We also examined M1 and M2 marker expression and the expression of key TME genes. Post the co-culture, we treated ovarian cancer cells with carboplatin and elucidated the function of programmed death–ligand 1 (PD-L1) in carboplatin chemoresistance. We investigated CD68 and PD-L1 expression in normal and cancerous ovarian tissues using immunohistochemistry (IHC). Finally, we analyzed the association between CD68 or PD-L1 expression and survival outcomes. Inducible nitric oxide synthase (iNOS) was downregulated, while the gene expression of M2 macrophage markers was increased in ovarian cancer cells. PD-L1, vascular endothelial growth factor (VEGF), Interleukin (IL)-6, IL-10, IL-12, signal transducer and activator of transcription 3 (STAT3), B-cell lymphoma 2 (BCL2), multidrug resistance 1 (MDR1), and colony stimulating factor 1 (CSF-1) were upregulated. Notably, PD-L1 was upregulated in both the ovarian cancer cells and macrophages. Ovarian cancer cells co-cultured with macrophages exhibited statistically significant carboplatin resistance compared to single-cultured ovarian cancer cells. PD-L1 silencing induced chemosensitivity in both types of co-cultured ovarian cancer cells. However, IHC results revealed no correlation between PD-L1 expression and patient survival or cancer stage. CD68 expression was significantly increased in cancer cells compared to normal or benign ovarian tumor cells, but it was not associated with the survival outcomes of ovarian cancer patients. Our study demonstrated that ovarian cancer cells interact with macrophages to induce the M2 phenotype. We also established that PD-L1 upregulation in both ovarian cancer cells and macrophages is a key factor for carboplatin chemoresistance.


Introduction
Epithelial ovarian cancer is the leading cause of cancer-related mortality in women with gynecologic cancers. In 2020, an estimated 313,959 new cases of ovarian cancer and 207,252 ovarian cancer-related deaths were reported worldwide [1]. Current standard treatments for ovarian cancer are based on cytoreductive surgery followed by platinum-based chemotherapy, such as carboplatin. Although many patients respond to initial chemotherapy, intrinsic or acquired chemoresistance remains a major challenge.
Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells in the tumor microenvironment (TME). Compelling evidence has suggested that TAMs promote key processes associated with tumor progression, metastasis, angiogenesis, and immunosuppression in various malignancies, including breast, ovarian, hepatocellular, and gastric cancers [2][3][4][5]. There are two distinct macrophage polarization states based on their activation-classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) [6]. TAMs in the TME have a predominantly M2-like phenotype in response to interleukin (IL)-6 and macrophage colony-stimulating factor (M-CSF) [7,8]. M2 macrophages in ovarian tumors have been confirmed by several studies, demonstrating a positive correlation between their high levels in tumors and the low overall survival of patients [9][10][11]. Chemotherapeutic agents, such as cisplatin and carboplatin, increase the potency of tumor cell lines to induce the M2 macrophage phenotype; this is possibly an indirect mechanism of chemoresistance [12].
Programmed death-1 (PD-1) is an immune checkpoint receptor that is upregulated in activated T-cells for immune tolerance. However, tumor cells overexpress the PD-1 substrate programmed death-ligand 1 (PD-L1) to suppress the immune system. It is expressed in both cancer cells and macrophages. The upregulation of PD-L1 expression in non-small cell lung cancers (NSLC) promotes a chemoresistance response [13]. Although PD-L1 expression in tumor cells is related to chemoresistance, little is known about the role of PD-L1 in the interaction between tumor cells and macrophages in the TME. An understanding of the regulatory mechanisms involved in PD-L1 expression, especially TAM-induced regulation in the TME, will be helpful in clinical treatment.
In this study, we investigated PD-L1 expression and the interaction between macrophages and tumor cells using the Transwell co-culture assay. We examined the role of M2 macrophage polarization and PD-L1 expression in carboplatin chemoresistance associated with ovarian cancer cells. We also investigated the relationship between TAM infiltration or PD-L1 expression and patient survival using immunohistochemical (IHC) analysis.

Cell lines and culture
We obtained the human ovarian cancer cell line SKOV3, human monocyte cell line THP-1, mouse ovarian cancer cell line ID8, and mouse macrophage cell line RAW264.7 from the Korean Cell Line Bank (Seoul, Korea). We cultured these cells in an RPMI 1640 medium (WELGENE, Gyeongsan, Korea), containing 10% fetal bovine serum (FBS; YounginFrontier, Seoul, Korea) and 1% antibiotic solution (ABS; Corning Inc, NY, CA, USA), at 37˚C in a humid atmosphere of 5% CO 2 . Moreover, we induced the THP-1 cells to differentiate into macrophages by adding 320 nM of 12-O-tetradecanoylphorbol-13-acetate (PMA) to the culture medium. Green fluorescence protein (GFP) was transfected into the SKOV3 cell line using a pCDH vector, and the transfected cells were selected with 3 μg/mL of puromycin.

Patients and tumor specimens
We obtained tumor samples from 47 patients with ovarian cancer and 35 patients with benign ovarian tumor, confirmed pathologically, who underwent surgery at Soonchunhyang University Cheonan Hospital. We used an additional seven normal ovarian tissue samples from our institution and 38 tumor samples from the Human Material Bank in Gangnam Severance Hospital. Ethical approval for this study was obtained from the Institutional Review Board (SCHCA2016-02-023). Informed written consent was acquired from all the patients.

Immunohistochemical staining of PD-L1 and CD68
We assessed PD-L1 expression via immunohistochemical staining. We sectioned 4 μm thick paraffin-embedded blocks of patient tissues, dried the slides for one day, and kept them at 60˚C for an hour. Antigen retrieval was performed using 3% H 2 O 2 and a 95˚C antigen retrieval buffer. The tissue sections were permeabilized with a 0.2% Triton solution and blocked with 5% bovine albumin serum in PBS for 15 min. They were then incubated overnight with a rabbit polyclonal primary PD-L1 antibody (1:100, Thermo Fisher, PA5-20343) at 4˚C. Thereafter, the sections were incubated with goat anti-rabbit IgG (H+L) and horseradish peroxidasetagged secondary antibodies (1:100, Thermo Fisher, 31460) for 1 h at 23˚C. The sections were then stained with DAB (3,3'-diaminobenzidine, VECTORLABS, SK-4100) and counterstained with 50% hematoxylin for 30 s. The slides were subsequently dried at 37˚C for 1 h and mounted with the Eukitt1 Quick-hardening mounting medium (Sigma-Aldrich, 03989-100ML). The slides were interpreted twice by two trained pathologists under an optical light microscope (Leica microsystems DMi1, Wetzlar, Germany). The CD68 + sections were counted in normal, benign, and ovarian cancer tissues and classified into low and high subsets based on the median value of the CD68 + counts in ovarian cancer sections. The PD-L1 sections were quantified based on the percentage of stained cells as follows: 0 staining intensity, no stained cancer cells; +1, <33% positively stained cancer cells; +2, 33-66% positively stained cancer cells; and +3, >66% positively stained cancer cells.

Wound healing assay
The wound healing capacity of the cells was measured using culture inserts (Ibidi, 81176). We cultured 1×10 5 /200 μL of ID8 cells in culture inserts for 24 h. Subsequently, we created a cellfree gap of 500 μm by removing the culture inserts. The old medium was removed, and cells were either treated with a co-culture (to recreate the TME) or a single-culture soup. The optical images were visualized after 0, 8, 16, and 24 h of wound creation. The percentage of wound closure fields was calculated using the Image J software (NIH, 1.52a).

Reverse transcription-polymerase chain reaction (RT-PCR)
We seeded and incubated 1×10 4 ID8 (or SKOV3) cells/well into 24-well plates and 1×10 4 RAW264.7 (or THP-1) cells onto polycarbonate Transwell inserts in 24-well plates for 72 h. Subsequently, we removed the supernatants and transferred the inserts to a new 24-well plate. We added 1 mL RiboEx (Geneall, 305-101, Seoul, Korea) to each well, according to the manufacturer's instructions, and incubated the plate for 5 min at room temperature. Next, we added 200 μL chloroform per 1 mL of RiboEx. This mixture was shaken vigorously for 15 s, incubated for 2 min at room temperature, and centrifuged at 12,000 × g at 4˚C for 15 min. We transferred the aqueous phase to a fresh tube and added 1 volume of RB1 Buffer (Geneall) to it; thereafter, we mixed the contents thoroughly by inverting the tube. Subsequently, we transferred up to 700 μL of the mixture to a mini-column type F and centrifuged it at �10,000 × g for 30 s at room temperature. We added 500 μL of SW1 Buffer (Geneall) to the mini-column and centrifuged it at �10,000 × g for 30 s at room temperature. Next, we added 500 μL RNW Buffer (Geneall) to the mini-column and centrifuged it at �10,000 × g for 30 s at room temperature. We then centrifuged it at �10,000 × g for an additional 1 min at room temperature to remove the residual wash buffer. We transferred the mini-column to a new 1.5 mL microcentrifuge tube and added 50 μL nuclease-free water to the center of the mini-column membrane. The column was kept stationary for 1 min and then centrifuged at �10,000 × g for 1 min at room temperature. The extracted RNA was immediately reverse-transcribed to cDNA using the ReverTra Ace1 qPCR RT Master Mix kit (TOYOBO, FSQ-201). Quantitative RT-PCR was performed using the SYBR Green Real-time PCR Master Mix kit (TOYOBO, QPK-201); the primer pairs are listed in Table 1. The PCR cycle included one cycle at 95˚C for 1 min, followed by 32 cycles at 95˚C for 15 s, 60˚C for 15 s, and 72˚C for 25 s. We used the Quantitative RT-PCR protocol from the CFX96™ System (BIO-RAD, 3600037). The PCR cycle included one cycle denaturation at 95˚C for 1 min, followed by 32 cycles at denaturation 95˚C for 15 s, primer annealing 60˚C for 15 s, and elongation 72˚C for 25 s.
We incubated 1×10 6 THP-1 cells per T75 culture flask for 24 h. Subsequently, the medium was changed to a serum-free RPMI 1640 medium containing 320 μM PMA (Merck, Germany), and the cells were cultured for 72 h to induce polarization. Thereafter, the medium was changed to an RPMI 1640 medium containing 10% FBS and 1% ABS, and the cells were cultured for 24 h. This medium was then changed to a serum-free RPMI 1640 medium, and the cells were cultured for a further 24 h. Thereafter, 30 μL of 3 μg/μL human PD-L1 siRNA (Sigma) was mixed with 90 μL transfection reagent and added to the cells.

Immunofluorescence staining and confocal microscopy
We reproduced the co-cultivation conditions using soup change. The co-culture soup was obtained from co-cultured T75 flasks (SPL Ltd., Pocheon, Korea) that were seeded with 5×10 5 SKOV3-GFP cells and 5×10 5 PMA-treated THP-1 cells. The single-culture soup was obtained from a single-cultured T75 flask that was seeded with only SKOV3-GFP cells. After three days, we collected the co-culture and single-culture soups from each T75 flask. Thereafter, we cultured 1×10 5 SKOV3-GFP and THP-1 cells/well in four-well chambered culture slides (SPL Ltd.) for 24 h. The old media was removed, and the cells were treated with co-culture and single-culture soups for 48 h. Thereafter, all the media was removed, and the cells were washed thrice with PBS and fixed using 4% formaldehyde for 15 min at room temperature. Following this, all the supernatants were removed, and the cells were blocked for 60 min with a solution containing 94.9% PBS, 4.8% FBS, and 0.3% Triton-X 100. The cells were incubated overnight with a rabbit polyclonal primary PD-L1 antibody (1:100, Thermo Fisher, PA5-20343) at 4˚C. Subsequently, the cells were incubated with Alexa Fluor1 Plus 594-tagged donkey anti-rabbit IgG (H+L) highly cross-adsorbed polyclonal secondary antibody (1:1000, Thermo Fisher, A32754) for 1 h at room temperature. We then performed Hoechst staining and compared the relative fluorescence intensities obtained under different culture conditions under a confocal laser scanning microscope (ZEISS LSM 710, ZEISS, Berlin, Germany).

Cell viability test after carboplatin treatment
We seeded 1×10 4 ID8 (or SKOV3) cells/well in 24-well plates and RAW264.7 (or THP-1) cells onto polycarbonate Transwell inserts in 24-well plates. We evaluated the effect of co-culturing Table 1. RT-PCR primer pairs designed based on sequence information obtained from the Ensembl genomic database (ensembl.org).

Western blotting
Cells were washed in phosphate-buffered saline (PBS) and lysed in Pro-Prep™ protein extraction solution (iNtRON, Seongnam, Korea). The protein concentration was determined by the bicinchoninic acid (BCA) assay using an xMark Microplate Absorbance 208 Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The lysate was centrifuged and the protein in the supernatant was denatured by boiling for 10 min at 100˚C. Equal quantities of protein (30 μg/lane) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was then blocked for 1 h in 5% skim milk. One membrane was incubated overnight at 4˚C with anti-human B-actin polyclonal antibody (Santa Cruz Biotechnology, sc-47778) diluted 1:500 and incubated with the secondary antibody for 2 h at room temperature. The other membranes were incubated overnight at 4˚C with anti-human PD-L1 polyclonal antibody (Thermo Fisher, PA5-20343) diluted 1:500 and incubated with the secondary antibody for 2 h at room temperature. The signal was detected using ECL western detection reagents (Advansta, Menlo Park, CA, USA) and a Molecular Imager Chemi-luminescence Bioimaging Instrument (Cellgenetek, Deajeon, Korea).

Statistical analysis
The wound closure percentage was statistically analyzed using the Student's t-test and the Statistical Package for the Social Sciences v19.0 (SPSS; IBM, Chicago, IL, USA). However, the rest of the data were analyzed using SPSS v23.0. The t-test was used to compare the expression of CD68 between normal or benign and cancer tissues. Survival analyses were performed using the Kaplan-Meier method, and differences between groups were tested using the log-rank test. Progression-free survival (PFS) was defined as the time interval between the surgery and tumor recurrence or the last follow-up. Overall survival (OS) was defined as the time interval between the diagnosis of the disease and death or the last follow-up. These independent prognostic factors, i.e., PFS and OS, were examined via multivariate analysis using the Cox regression model. Differences were considered statistically significant at p-values < 0.05.
Recurrence was observed in 35 patients (41%), and the median value for progression-free survival was 20.48. In all patients, the median overall survival was 51.49.

Influence of macrophages on tumor cells
We investigated the effects of macrophages on cell migration by performing an in vitro wound healing assay. Our results reveal that the co-cultured ID8 cells fill up the wound space faster than the single-cultured ID8 cells. This indicates that co-culture improves the migration and proliferation of ID8 cells. We further deduce that macrophages pose challenges in the elimination of cancer cells (Fig 1).

Macrophages differentiate into pro-tumorigenic M2 phenotypes after coculture with tumor cells
We analyzed the mRNA extracted from macrophages co-cultured with and without tumor cells via RT-PCR. The co-cultured macrophages exhibit upregulation of vascular endothelial cell growth factor (VEGF), signal transducer and activator of transcription 3 (STAT3), PD-L1, CD206, IL-10, and ARG1. Notably, these genes are immunosuppressive and pro-tumor markers in M2 macrophages. However, IL-12 is upregulated in M1 phenotypes (Fig 2A).

Macrophages induce tumor cells to express immunosuppressive molecules
RT-PCR was used to analyze mRNA expression in tumor cells co-cultured with or without macrophages. The co-cultured ovarian cancer cells show increased expression of PD-L1, VEGF, STAT3, IL-10, IL-6, B-cell lymphoma 2 (BCL2), and MDR1. These genes are related to immunosuppression, chemoresistance, and angiogenesis ( Fig 2B). Confocal microscopic images indicate that the expression of PD-L1 is markedly increased in both ovarian cancer cells and macrophages after co-culture (Fig 2C-2F).

Macrophages induce chemoresistance against carboplatin in tumor cells
Cell viability results reveal that the chemosensitivity of ovarian cancer cells is reduced after coculturing with macrophages. Moreover, we observe a significant difference in the chemosensitivity between 100 and 200 μM carboplatin concentrations (Fig 3).

Silencing of PD-L1 in both macrophages and tumor cells reverses chemoresistance to carboplatin
Treatment with PD-L1 siRNA reduces the mRNA expression of PD-L1 to 80.48% in SKOV3 cells and to 65.46% in THP-1 cells (Fig 4A). PD-L1 siRNA also reduces the PD-L1 protein expression in SKOV3 cells and THP-1 cells (Fig 4B). Furthermore, PD-L1 silencing increases the chemosensitivity of SKOV3 cells to carboplatin (p < 0.001). Increased chemosensitivity to carboplatin is the highest when PD-L1 is silenced in both the SKOV3 and THP-1 cells (p < 0.001). PD-L1 silencing in THP-1 cells alone is not statistically significant (p = 0.472) ( Fig  4C). Next, we labeled ovarian cancer cells with GFP to evaluate their chemosensitivity. The intensity of GFP significantly decreases post-PD-L1 silencing (Fig 4D).

Association between PD-L1 expression and the OS and PFS of ovarian cancer patients
We assessed PD-L1 expression in the tumor specimens of 85 ovarian cancer patients via IHC. Amongst these patients, 67 (79%) had PD-L1 low-positive tumors and 18 (21%) had PD-L1 high-positive tumors. No statistically significant association is observed between PD-L1 expression and the survival of both groups. Thus, we infer that PD-L1 expression is not a prognostic factor for ovarian cancer (Fig 5).

Upregulation of CD68 in ovarian cancer cells and its association with OS and PFS
We compared the expression of CD68 in 85 cancer, 7 normal, and 35 benign ovarian tissues and found that the immunohistochemistry activity of CD68 is higher in the ovarian cancer tissues compared to that in the others. Tissues were divided into low and high groups based on the median value of CD68 expression in ovarian cancer tissues. The relationship between CD68 expression and survival outcomes was determined (Fig 6). No statistically significant association is observed between CD68 expression and the survival of both groups.

Discussion
The primary reason for poor survival rates in ovarian cancer patients is the resistance to chemotherapeutic agents such as carboplatin and paclitaxel. Although the mechanism of chemoresistance in ovarian cancer is not completely understood, several studies have reported that factors such as the TME regulate this mechanism. In fact, the interplay between tumor cells and TAMs in the TME has been proposed as one of the reasons for therapeutic failure in cancer patients. The invasiveness of ovarian cancer cells is induced by various cytokines such as IL-10 and IL-6, which are secreted by the TAMs that have M2-like phenotypes and express CD206 and arginase-1 (ARG1). We determined that TAMs had increased expression of IL-10, IL-6, CD206, and ARG1, whereas ovarian cancer cells had increased expression of IL-10, IL-6, VEGF, STAT3, and PD-L1. Moreover, the co-cultured ovarian cancer cells exhibited faster migration and proliferation in the wound healing assay than the single-cultured ovarian cancer cells. The key pro-inflammatory factor IL-6 has been implicated to be involved in the tumor progression and MDR of various cancers [14][15][16]. It induces MDR by increasing the expression of MDR-1 and the apoptosis inhibitory protein Bcl-2. Furthermore, we previously found that certain cytokines and transcriptional factors, including IL-6, IL10, IL-27, STAT, and NF-kB, regulate PD-L1 expression in various cancers [17][18][19][20][21]. Macrophages activate STAT3 in tumor cells by expressing IL-6, thereby enhancing the proliferation and survival of malignant cells even during chemotherapeutic treatment [22]. We demonstrated that IL-6, MDR-1, and BCL2 increased expression in the ovarian cancer cells co-cultured with macrophages. The proteins PD-1 (encoded by PDCD1) and PD-L1 (encoded by CD274) are crucial factors for immune homeostasis [23]. However, the PD-1/PD-L1 axis in the TME is deregulated by cancer cells and TAMs to escape immune surveillance [24]. Therefore, PD-L1-expressing TAMs contribute to an immunosuppressive TME. We identified the robust expression of PD-L1 in both the tumor cells and TAMs following co-culture. Remarkably, our Transwell coculture assay revealed that ovarian cancer cells became resistant to carboplatin after co-cultivation with macrophages. Although the role of the PD-L1 and PD-1 interaction in regulating Tcell suppression has been well established, little is known about PD-L1 signaling in ovarian cancer chemotherapy resistance. Thus, we hypothesized that PD-L1 in both ovarian cancer cells and TAMs might be associated with carboplatin resistance. In our experiments, PD-L1 silencing in SKOV3 cells increased their chemosensitivity to carboplatin. Moreover, when PD-L1 was silenced in both SKOV3 and THP-1 cells, the chemosensitivity to carboplatin was the highest. Therefore, we concluded that PD-L1 knockdown in both ovarian cancer cells and macrophages increased the sensitivity of the ovarian cancer cells to carboplatin. Although blocking the PD-1 and PD-L1 axis is an attractive target for cancer treatment, many preclinical and clinical studies have concentrated on the relationship between PD-L1 and immunosuppression, but not chemoresistance. It has been reported that microRNA-197 enhances PD-L1 expression and chemoresistance in lung cancer cells [25]. Xu et al. (2016) indicated that micro-RNA-424 treatment reduces PD-L1 expression by directly binding to the 3 0 -untranslated region of human and mouse genes and reverses chemoresistance via the T-cell immune response [26]. Furthermore, Zhang, et al. [13] reported that alterations in PD-L1 levels can regulate chemoresistance in lung cancer. They also reported that upregulation of PD-L1 possibly occurs via the activated PI3K/AKT signaling pathway [27].
While PD-L1 expression is associated with poor patient prognosis for a variety of solid tumors, including cervical cancer, its prognostic value in ovarian cancer remains controversial. Wang [32] performed a meta-analysis of 12 studies, involving 1630 ovarian cancer patients, by conducting an IHC analysis of PD-L1 expression to investigate the prognostic impact of PD-L1 in ovarian cancer. Wang suggested that PD-L1 expression is not linked to the tumor grade, clinical stage, lymph node status, tumor histology, and patient OS and PFS [32]. Our results are consistent with these observations, as we did not observe any association between PD-L1 expression and PFS or OS. Our study indicates that interactions between ovarian cancer cells and macrophages confer ovarian cancer cells with migratory, proliferative, and chemotherapy-resistant properties. In contrast, macrophages polarize to the M2 phenotype following co-culture. Nonetheless, both tumor cells and macrophages show robust PD-L1 expression upon co-culture. However, PD-L1 knockdown reverses the chemoresistance of ovarian cancer cells to carboplatin.
This study has the limitation of a small sample size. Additionally, the expression of PD-L1 in ovarian cancer tissues was not found to have any association with patient survival. However, our study offers a possible mechanism for PD-L1-related chemoresistance in ovarian cancer cells and macrophages. Further research is required to completely elucidate this mechanism for the discovery of novel therapeutic strategies.